The amplitudes and durations of cardiac action potentials are largely determined by voltage-gated K+ (Kv) channels, and in most cardiac cells, multiple Kv currents with distinct time- and voltage-dependent properties are co-expressed. Important insights into the potential molecular basis of functional myocardial Kv channel diversity were provided with the identification of large numbers of Kv channel pore-forming (1) and accessory (2) subunits. In addition, accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising (four) Kv 1 and multiple Kv2 subunits and regulatory proteins, although the roles of accessory and regulatory proteins in controlling the functional cell surface expression and the properties of myocardial Kv channel are poorly understood. This new R21 proposal will test the novel hypothesis that voltage-gated Na+ (Nav) channel accessory (Nav2) subunits regulate the functional expression of Kv4-encoded fast, transient outward Kv (Ito,f) channels in ventricular myocytes rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. This hypothesis reflects recent biochemical findings demonstrating that the Nav21 (SCN1b) and Nav22 (SCN2b) subunits co- immunoprecipitate with Kv4 1 subunits in native Kv4-encoded Kv channel macromolecular protein complexes. There are two related aims in this proposal, and these will be pursued in parallel. Specifically, the studies here will test the novel hypothesis that Nav21 functions to regulate the cell surface expression and/or the properties of Kv4-encoded myocardial Ito,f channels (aim #1) rather than, or in addition to, regulating Nav channels (aim #2) and determine directly the role of Nav21 in shaping myocardial action potential waveforms.(aim #2). To achieve these aims, the expression of the endogenous Nav21 subunit will be manipulated in (mouse) ventricular myocytes in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of Ito,f (and Nav) channels will be determined. Parallel experiments will be completed on myocytes isolated from mice (Scn1b-/- ) harboring a targeted disruption of the Scn1b (Nav21) locus. It is anticipated that these studies will provide new and fundamentally important insights into the mechanisms that control the expression and the functioning of macromolecular Kv channel complexes. In addition, the results of the studies here will guide future investigations focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of myocardial membrane excitability and in the derangements in cardiac excitability linked to mutations in SCN1b. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels control the heights and durations of myocardial action potentials and contribute importantly the generation of normal cardiac rhythms. Changes in Kv channel expression and/or properties are observed in a number of inherited and acquired cardiac diseases, and these changes can have profound physiological consequences, including increasing the risk of potentially life-threatening cardiac arrhythmias. Although accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising pore-forming (1) subunits and a variety of accessory (2) subunits that affect channel stability, trafficking and/or properties, very little is presently known about the roles of accessory subunits in the physiological regulation of Kv channels in cardiac myocytes. Exploiting molecular genetics strategies to manipulate channel subunits in vivo and in vitro, this new research program is focused on defining the physiological role(s) of the Nav2 (SCNxb) accessory subunits in regulating the excitability of cardiac myocytes and on testing the novel hypothesis that the Nav2 accessory subunits function to regulate Kv channels rather than, or in addition to, regulating voltage- gated Na+ (Nav) channels. These studies will provide new and fundamentally important insights into the physiological roles of Nav2 subunits in the myocardium and into the molecular mechanisms controlling myocardial membrane excitability.